Human adenoviruses have long been recognized as pathogens, causing a broad spectrum of diseases. Most commonly, adenoviruses cause respiratory diseases. Depending on the infecting serotype, they may also cause various other diseases, such as gastroenteritis, conjunctivitis, rash and cystitis. Symptoms of respiratory illness caused by adenovirus infection include the common cold syndrome, pneumonia, croup and bronchitis. Patients with compromised immune systems are especially susceptible to severe complications of adenovirus infection. Acute respiratory disease (ARD) can be caused by adenovirus infections during conditions of crowding and stress.
Since a high number of viral and bacterial pathogens can cause similar symptoms, accurate identification of the etiologic agent is very important for specific classification and differentiation as well as for subsequent treatment.
Adenoviruses are medium-sized (90-100 nm), non-enveloped icosohedral viruses containing double-stranded DNA. There are 49 immunologically distinct serotypes which are classified in 6 different subtypes (A through F) that can cause human infections.
Although epidemiologic characteristics of the adenoviruses vary by type, all are transmitted by direct contact, faecaloral transmission, and occasionally waterborne transmission. Some adenoviruses (e.g. serotypes 1, 2, 5, and 6) have been shown to be endemic in parts of the world where they have been studied, and infection is usually acquired during childhood.
Acute respiratory disease is most often associated with adenovirus types 4 and 7 in the United States. For some adenovirus serotypes, the clinical spectrum of disease associated with infection varies depending on the site of infection; for example, infection with adenovirus 7 acquired by inhalation is associated with severe lower respiratory tract disease, whereas oral transmission of the virus typically causes no or mild disease. Outbreaks of adenovirus-associated respiratory disease have been common in the late winter, spring, and early summer.
Several methods have been established for the identification of adenovirus infections. While conventional methods such as visualization by electron microscopy, viral culture or agglutination assays with antibody-coated beads are tedious and time-consuming, other methods such as dot blot hybridization lack a sufficiently high sensitivity and are not amenable to high-throughput. Therefore, detection assays have been developed which are based on polymerase chain reaction (PCR). For example, real-time PCR methods for detecting and quantifying the adenoviral load in immunosuppressed patients have been established (Leurez-Ville et al. 2004, Clin. Infect. Dis., 38: 45-52). Multiplex real-time PCR methods for the detection of adenoviruses have also been described (Gu et al., 2003, J. Clin. Microbiol. 41: 4636-4641). One problem encountered in the detection of adenoviruses via PCR resides in the high sequence diversity between different adenovirus subtypes and serotypes which results in a high degree of mismatching of viral DNA with primers and probes which are aimed at the detection of all subtypes in a biological sample. As a consequence, sensitivity and specificity of the PCR methods described in the prior art is low.
The problem underlying the present invention is the provision of primers and probes that allow for the reliable detection of essentially all adenovirus serotypes in a sample which are involved in respiratory infections, in particular serotypes 1, 2, 3, 4, 5, 6, 7, 11, 14, 16, 21, 34, and 35. Theses adenovirus serotypes belong to the subtypes B, C and E. The primers and probes should in particular be suitable for use in a multiplex PCR approach for the parallel detection of different viral and/or bacterial pathogens.